ADP is an important agonist for platelet activation and plays a major role in hemostasis and thrombosis. Given its critical role in platelet function, the molecular mechanisms of ADP-mediated physiological processes warrant a better understanding. During the previous grant periods, we have demonstrated the presence of three ADP receptor subtypes, P2Y1, P2Y12, and P2X1 on platelets and elucidated several signaling mechanisms in platelets. Here, we propose to enhance our understanding by further elucidating the molecular mechanisms of ADP-induced platelet activation. We hypothesize that diverse signaling cascades downstream of the P2Y receptors play a crucial role in platelet activation, and that specific protein kinase C and PI-3 kinase isoforms contribute significantly to these events. We propose to test this hypothesis by pharmacological, biochemical, and molecular genetic approaches. 1) We hypothesize that specific isoforms of protein kinase C (PKC) are activated downstream of the Gq-coupled P2Y1 receptors and regulate platelet function. We hypothesize that ADP activates other novel PKC isoforms that have a functional role in the platelet activation. In our preliminary studies we have shown that ADP activates PKC? through the P2Y1 receptor that regulates ADP-induced platelet functional responses. 2) We propose to characterize the mechanism by which PKC? regulates ADP-induced platelet activation. We hypothesize that PKC? phosphorylates and negatively regulates the Gai class of proteins in platelets. 3) We hypothesize that different PI-3 kinase isoforms [unreadable], ?, and d, downstream of the P2Y12 receptor, play distinct roles in platelet activation. We propose to investigate the role of PI-3 kinases in ADP-induced functional responses including fibrinogen receptor activation, potentiation of dense granule release, and thromboxane generation, as well as ADP-induced phosphorylation of Akt, Erk, and PLA2. 4) We hypothesize that distinct intracellular domains of P2Y1 and P2Y12 receptors couple to the Gq and Gi proteins. This hypothesis will be tested by the molecular and cell biological approaches to identify the G protein-coupling domains on the P2Y1 and P2Y12 receptors. Continuing on our progress in the last grant period on this aim, we will continue to test this hypothesis by deletion mutagenesis, site directed mutagenesis, and chimeric receptor approaches. We have strong preliminary data supporting each of the above specific aims. These studies will enhance our understanding of the signaling pathways downstream of ADP receptors and their role in platelet activation, and might identify potential newer targets for the treatment of thrombosis. PUBLIC HEALTH RELEVANCE: ADP plays a significant role in the regulation of platelet function through its cell surface receptors. Platelet activation is critical for hemostasis and can lead to thrombotic events. One of the very successful anti-thrombotic drugs, clopidogrel, elicits its effects through blocking the platelet P2Y12 receptors. Thus, signal transduction mechanisms downstream of ADP receptors are important to understand the molecular basis of platelet activation. The proposed research examines the regulation and function of signaling events downstream of ADP receptors through a combination of biochemical, pharmacological and genetic approaches. An in-depth understanding of these mechanisms will aid in identifying novel targets of antithrombotic therapeutics. [unreadable] [unreadable] [unreadable] [unreadable]